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Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization

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posted on 2023-02-17, 13:06 authored by Molly J. Kirk, Arya Gold, Ashvin Ravi, Gabriella R. Sterne, Kristin Scott, Evan W. Miller
Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.

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